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Abstract

The in vitro cell tests and in vivo animal tests were performed to investigate the feasibility of the photothermal therapy
based on porous silicon (PSi) in combination with near-infrared (NIR) laser. According
to the Annexin V- fluorescein isothiocyanate Apoptosis assay test results, the untreated
cells and the cells exposed to NIR laser without PSi treatment had a cell viability
of 95.6 and 91.3%, respectively. Likewise, the cells treated with PSi but not with
NIR irradiation also had a cell viability of 74.4%. Combination of these two techniques,
however, showed a cell viability of 6.7%. Also, the cell deaths were mostly due to
necrosis but partly due to late apoptosis. The in vivo animal test results showed that the Murine colon carcinoma (CT-26) tumors were completely
resorbed without nearly giving damage to surrounding healthy tissue within 5 days
of PSi and NIR laser treatment. Tumors have not recurred at all in the PSi/NIR treatment
groups thereafter. Both the in vitro cell test and in vivo animal test results suggest that thermotherapy based on PSi in combination with NIR
laser irradiation is an efficient technique to selectively destroy cancer cells without
damaging the surrounding healthy cells.

Introduction

In recent years, photothermotherapy (PTT) techniques based on inorganic nanomaterials
and near-infrared (NIR) light have attracted significant attention owing to their
advantages over conventional surgical treatments. The advantages of PTT include the
anticipated reduction in morbidity and mortality, low cost, suitability for real-time
imaging guidance, and the ability to perform ablative procedures on outpatients because
of its non-invasive nature [1]. In the conventional PTTs based on simple heating, i.e., hyperthermia [[2], and references therein], most treatment failures result from insufficient temperature
rises in the tumor tissues. Therefore, it is essential to use a thermal coupling agent
with a good photothermal property to secure irreversible destruction of tumor cells
in a short time without damaging adjacent healthy cells in thermotherapy.

The inorganic nanomaterials currently demonstrated as thermal coupling agents in PTTs
are gold nanoparticles (Au NPs) [3-12], gold nanorods [13-16], gold nanoshells [1,17-19], gold nanocages [20,21], gold nanocrystals [22,23], single wall carbon nanotubes (SWCNTs) [24-26], and porous silicon (PSi) [27,28]. Of these nanomaterials, we are particularly interested in PSi because it is known
to have many important properties such as biocompatibility [29], biodegradability [30,31], and a readily functionalized surface [32] which a therapeutic agent should have desirably as well as an excellent photothermal
property [33]. In addition, PSi has a merit that it can be easily prepared by the simple electrochemical
anodization of silicon. As regards biomedical applications of PSi, their use in drug
delivery [34-39] and photodynamic therapy [40-42] applications have been reported before. We previously reported on the excellent heat
generation ability of PSi and the ability of PSi to irreversibly destroy cancer cells
under NIR laser irradiation by using temperature rise measurement and MTT assay results,
respectively [27,28]. In this paper, we report the Annexin V-fluorescein isothiocyanate (FITC) apoptosis
assay test and in vivo animal test results of PSi in combination with NIR laser to investigate the ability
of PSi to kill cancer cells as well as the death modes of cancer cells and the ability
of PSi to inhibit the growth of tumors, respectively.

Experimental details

Preparation of PSi/EtOH:PEG drug solutions

First, meso-PSi layers were prepared on 2.5 cm × 2.5 cm × 0.05 cm pieces of p-type
Si(100) with a resistivity of 1-5 mΩcm by anodic etching in an 3:1 (by volume) solution
of 46% HF and 95% C2H5OH at a current density of 200 mA/cm2 for 150 s. PSi is generally classified into three different types in terms of the
pore size: macro-PSi (d > 50 nm), meso-PSi (2 nm < d < 50 nm), micro-PSi (d < 2 nm). According to our experience, meso-PSi is the most suitable for photothermotherapy
since it shows the highest photothermal effect and can be easily to be fractured into
nanoparticles with proper sizes. The porosity and thickness of the PSi layers determined
by weight measurements [43] were about 73% and 55 μm, respectively. The details of the anodization process are
described elsewhere [27]. The PSi layers formed on Si(100) were then lifted off by anodic etching in an 1:15
(by volume) solution of 46% HF and 95% C2H5OH at a current density of 4 mA/cm2 for 250 s. Next, the free-standing PSi layers were fractured by ultrasonicating in
10 mL of ethanol for 24 h. The PSi nanoparticles were subsequently filtered twice
by using a 450 nm membrane first and then by using a 220 nm membrane. PSi/EtOH:PEG
drug solutions were prepared by dispersing the resulting PSi particles in 10 mL of
ethanol mixed with 10 mL of thiolated polyethyleneglycol (PEG-SH) and centrifuged
for 24 h until all the PSi particles were dispersed.

Measurement of heating of the PSi/EtOH:PEG solution by NIR irradiation

Heterochromatic NIR light irradiation was performed on six different samples by using
NIR laser. The samples include a PSi/EtOH:PEG solution and a EtOH:PEG solution as
well as a solid PSi (a free standing PSi layer). The three different kinds of samples
were irradiated continuously for 20 min by the NIR laser at 1.5 W/cm2. The distance between the laser source and each sample was fixed to be 2 cm. Change
in the temperature of the samples with the NIR exposure time was measured at 30-s
intervals by using an IR thermometer (model: AZ 8859, max. output: 1 mW, wavelength:
670 nm, measurement range: -20 to 420°C).

Annexin V- FITC apoptosis assays

CT-26 cells were cultured in DMEM. The incubations of 1 × 106 CT-26 cells were carried out at 37°C and in 5% CO2 atmosphere for approximately 24 h in 24-well plates, with the cells having been seeded
in a 100 nm dish for approximately 18 h before incubation. After incubation, the cell
media were removed from the wells, and the cells were washed using PBS and then incomplete
DMEM was added to each well. Then, the PSi/EtOH:PEG solution was added to each well.
Annexin V- FITC apoptosis assays were performed on four different mouse CT-26 cell
sample groups to see the ability of our technique to irreversibly destroy cells and
the modes of cell deaths: the CT-26 cell control group given neither PSi nor laser
treatment, the CT-26 cell group not treated with PSi but with laser, the group not
treated with laser but with PSi, the group treated with both PSi and laser. For the
preparation of the last sample group, CT-26 cells were treated with the PSi/EtOH:PEG
drug solution (0.7 g/L) first and then NIR laser at 600 mW/cm2 for 20 min. Next, the 2 × 106 cells were removed from the culture, washed twice with cold PBS, and double-stained
with Annexin V-FITC and propidium iodide (PI) (BD Biosciences, San Jose, CA, USA)
in Annexin-binding buffer, followed by analysis on a FAC-Scalibur flow cytometer (Becton
Dickinson, San Jose, CA, USA) equipped with a 488-nm argon laser. To avoid nonspecific
fluorescence from dead cells, live cells were gated using forward and side scatter.

Trypan blue cell death assays

Cell viability was performed by the trypan blue cell death assay. Briefly, CT-26 cells
were plated at a density of 3 × 105 cells in 60 mm culture dishes for 24 h. Then, the medium was removed, and the cells
were treated with a PSi/EtOH:PEG solution to each plate. The final concentration of
ethanol in the medium was ≤0.5% (v/v). Detached CT-26 cells treated with a PSi/EtOH:PEG
solution were exposed to NIR laser at 600 mW/cm2 for 20 min. After NIR irradiation, a collection of supernatants and adherent cells
obtained by trypsinization was incubated in 0.4% trypan blue and pipetted onto a hemocytometer
and manually counted under a microscope at ×100 magnification. The percentage of cells
admitting trypan blue dye to the total number of cells was determined by counting
three different fields for each experimental condition, which was done in triplicates
[44].

In vivo animal tests

Animal care and all experimental procedures were conducted in accordance with the
Guide for Animal Experiments edited by the Korean Academy of Medical Sciences. The
CT-26 cells (1 × 106 cells) were suspended in 100 μL PBS, and subcutaneously injected into the back of
male mice of each group (n = 5, 5- to 6-week-old, Balb/c). When the tumors were grown up to a volume of 65-70
mm3, mice were randomized into four groups: (a) mice were simply monitored without any
other treatment; (b) mice were intratumorally injected with 100 mL of PBS and then
irradiated NIR laser 4 times at 1.5 W/cm2 for 2 min each time with a time interval of 2 min under the ether anesthesia; (c)
a PSi/EtOH:PEG solution (0.7 g/L, 100 μL) was intratumorally injected without NIR
laser irradiation; (d) a PSi/EtOH:PEG (0.7 g/L, 100 μL) was injected into tumor, then
NIR laser was immediately irradiated on the tumor region in the same manner as in
(a). The mice were anesthetized by injecting 40 μL of a 9:1 solution of ketamine (100
mg/mL) and rompun (100 mg/mL). Tumor volumes, animal body weights, and tumor conditions
were recorded weekly for the duration of the study [45]. The tumor size of each group was measured using a skinfold caliper, and tumor volumes
were calculated using the following equation: tumor volume = ab2/2, where a is the maximum diameter of tumor and b is the minimum diameter of tumor [46]. All the procedures for in vivo experiments were performed in accordance with Inha University of Biomedical Science
guidelines on animal care and use.

Results and discussion

Photothermal properties of the PSi/EtOH:PEG solution

The PSi nanoparticles functionalized with PEG were well solublized, so that they were
uniformly distributed in the PSi/EtOH:PEG solution without forming any floating particles,
precipitates, or agglomerates for a long period of time as shown Figure 1a. Functionalization of PSi with PEG is necessary to enhance the internalization of
PSi particles into cells as well as the attachment of antibodies to PSi particles
for the systematic administration of cancer. Ethyl alcohol was also used to enhance
the dispersion of PSi nanoparticles in the solution. Figure 1b displays the size distribution of the PSi nanoparticles after being filtered by using
a 220 nm membrane, which was in a range from 80-220 nm in diameter with a mean diameter
of approximately 140 nm. The photothermal property was compared between solid PSi,
PSi/EtOH:PEG solution (0.7 g/L), and EtOH:PEG solution in Figure 2. The surface temperature of each sample during exposure to 808 nm NIR laser at 1.5
W/cm2 was measured by using an IR thermometer. The PSi nanoparticles exhibited a very rapid
increase in temperature for the first 1 min and a slow increase for the next 5-6 min.
The temperature reached at approximately 70°C in about 6 min upon NIR laser irradiation
and did not nearly changed thereafter. The net temperature rises shall have been 47°C,
taking the temperatures of the PSi film at the exposure time of 0 min (~23°C) into
consideration. This high photothermal effects in PSi is mainly attributed to the high
absorbance and the high surface-to-volume ratio due to the numerous micropores in
PSi [33]. The temperature of the PSi/EtOH:PEG solution changed with the NIR exposure time
in a pattern similar to the solid PSi, but the temperature was much lower than that
of the PSi nanoparticles presumably due to the absorption of heat by the liquid phases
such as EtOH and PEG in the solution. The temperature difference between PSi/EtOH:PEG
and EtOH:PEG is about 10°C for the NIR irradiation time larger than 15 min, which
may seem to be somewhat small for thermotherapy. In the in vivo animal tests, the EtOH:PEG solution injected directly into tumors would move easily
to the whole body of the mouse. Consequently, a very little amount of remnant solution
would stay in the tumors. In contrast, the PSi/EtOH:PEG solution injected directly
into tumors would stay in the tumors for a longer time because it has a higher viscosity
than the simple EtOH:PEG solution. Therefore, there would be a big difference between
the two solutions in the actual effect of destroying cancer cells.

Figure 1.PSi particle size distribution: (a) PSi/EtOH:PEG solution and (b) the distribution of the diameter of PSi nanoparticles in the PSi/EtOH:PEG solution.

Annexin V- FITC apoptosis assay tests

The fluorescent-activated cell sorter (FACS) flow cytometry profiles (Figure 3a-d) obtained as a result of Annexin V-FITC Apoptosis assay represent Annexin V-FITC
staining in X-axis and PI in Y-axis. The four sections of the quadrant in each profile from the upper left in a
clockwise direction represent necrosis, late apoptosis, early apoptosis, and live
cell, respectively. Of these four kinds of cell modes, necrosis and late apoptosis
are usually considered as cell death. The Annexin V-FITC Apoptosis assay results in
(Figure 3a-d) are summarized in Figure 3e. The untreated cells and the cells exposed to NIR laser without PSi treatment had
a cell viability of 95.6 and 91.3%, respectively. Likewise, the cells treated with
PSi but not with NIR irradiation also had a cell viability of 74.4%. Combination of
these two techniques, however, showed a cell viability of 6.7%, implying that most
cells are killed. The group treated with both PSi and NIR laser shows substantially
higher cell death (necrosis + late apoptosis) rate than those not given both treatments.
It can also be seen in Figure 3e that the cell deaths are mostly due to necrosis but partly due to late apoptosis.
This in vitro cell test result suggests that only combination of PSi and NIR laser treatments can
kill cells. The viability of 74.4% for the cells treated with PSi but not with NIR
irradiation seems to be low, suggesting that PSi is somewhat toxic. This toxicity
of PSi may originate from HF residues on the surfaces of the PSi nanoparticles due
to incomplete washing. More effort should be made to remove all the HF residues during
the course of PSi nanoparticles preparation.

We previously reported as a result of the in vitro cell test based on MTT assay that the cell viabilities of the mouse groups untreated,
treated only with PSi, treated only with laser, and treated with PSi followed by laser
treatment were 99.8, 95.2, 98.1, and 2.6%, respectively [28]. The present Annexin V-FITC Apoptosis assay result is somewhat worse than the previous
MTT assay result. In the previous test, the PSi/NaCl suspension was used instead of
the PSi/EtOH:PEG solution as a PSi drug solution. Another difference is that the PSi
concentration in the PSi/NaCl suspension (~10.0 g/L) was far higher than that in PSi/EtOH:PEG
solution (0.7 g/L) since the PSi was not filtered by using a 220-nm membrane in the
former suspension whereas PSi was filtered prior to the in vitro cell test in the latter solution. Therefore, the higher cell viability, i.e., the
lower cell death rate of the group treated with both PSi and laser in the present
test may be mainly attributed to the lower PSi concentration in the PSi drug solution.

Trypan blue cell death assay tests

To determine whether the effect of PSi nanoparticles under the NIR laser irradiation
on cells was cytotoxic, trypan blue cell death assay was performed on mouse CT-26
cells to investigate localized photothermal destruction of the cancer cells. High-magnification
optical microscopy images of the cells dispersed in the PSi/EtOH:PEG solution given
the laser treatment is shown in Figure 4. The cells were first treated with a 0.7 g/L PSi/EtOH:PEG solution. Next, the cells
were exposed to laser and then stained by trypan blue dye to examine cell damage.
The color of the dead cells usually turns black after staining treatment. It can be
seen in Figure 4 that more than 60% of the cells irradiated with NIR laser have turned black in color,
indicating cell death.

Figure 4.Trypan blue staining. Optical microscopic images of the CT-26 cells treated with a PSi/EtOH:PEG solution
(0.7 g/L) followed by NIR laser treatments (for 20 min at 600 mW/cm2) 4 times for 2 min each time with a time interval of 2 min. The cells were stained
using trypan blue dye after the NIR laser treatments to examine cell damage. The cells
indicated by arrows are some examples of dead cells turned blue after staining.

In vivo animal tests

The in vitro cell test results show the photothermal effect of the thermotherapy based on PSi combined
with laser on cell death, but it does not guarantee that the thermotherapy can inhibit
tumor growth. To confirm that the photothermal effect of PSi combined with NIR laser
could efficiently destroy tumor cells without giving damage to surrounding healthy
cells, we attempted in vivo therapeutic examinations against Balb/c mice bearing (CT-26) on their backs. When
the tumors were grown up to a volume of approximately 100 mm3, PSi/EtOH:PEG solution (0.7 g/L, 100 μL) were then injected directly into the tumor
regions. A mouse ready for the irradiation was located under the focal lens through
which NIR laser could be focused to have a power density of 1.5 W/cm2 and irradiated 4 times for 2 min each time with a time interval of 2 min. This intermittent
laser irradiation was designed to minimize the damage of the healthy tissues adjacent
to the tumor tissues. As shown in Figure 5, the mouse treated with a PSi/EtOH:PEG solution and NIR laser irradiation shows perfect
tumor destruction 25 days after the treatments. It appears that the tumor has shrinked
to almost zero volume at day 5 post-treatment. The tumescent part formed on the laser-treated
region is not the shrinked tumor but looks like a kind of water blister formed by
thermal energy from PSi nanoparticles. Finally, the tumor has completely disappeared
at day 25 post-treatment although the complete disappearance of the tumor is not clearly
observable owing to the regrown fur. Comparison of the tumor site between the four
groups treated differently, however, more clearly indicates the complete destruction
of the tumor in the group treated both PSi and laser. It is worthy of noting that
the surface structures including the epidermis and subcutaneous tissue got no damage
such as carbonization at all. It is common that an esker or a black skin burn mark
forms and it falls off many days after treatment in PTT. Even no such marks were observed
on the mice in the group treated both PSi and laser. The intact surface structures
suggest that the treatment parameters including the power intensity of the laser and
the PSi concentration of the PSi/EtOH:PEG solution was adequate. It is well known
that the degree of damage of the surface structures in PTT strongly depends on the
treatment parameters. The mice given both PSi and laser treatments remained healthy
without any recurrence of tumors and side effects for more than 3 months. In contrast,
the other three groups including the control group, the group treated only with PSi,
and that treated only with NIR laser show significant growth of tumors after the treatment.
The size of the tumors in these groups is in a range of 1.1-1.5 cm at day 5 post-treatment.
The tumors grew continuously to be 1.8-2.7 cm in diameter at day 25 post-treatment.
The same kind of in vivo animal tests were repeated for another two sets of mouse
groups and similar results were obtained, implying that these results are very reproducible.
Figure 6 compares the tumor growth rate between the four different experimental mouse groups.
The tumors in the mice treated with both PSi and laser show almost zero volume change,
whereas the tumors in the other three groups continued to grow until they died (Table
1).

Figure 6.Change of tumor volume. Tumor volume (a) and ratio volume (b) of CT-26 tumor cell xenografts. Tumor volumes were measured once a week after sample
treatments. The group treated with a PSi/EtOH:PEG solution followed by NIR laser treatments
(4 times for 2 min at 1.5 W/cm2 each time with a time interval of 2 min) shows efficient tumor growth inhibition compared
with other experimental groups.

Weight change after laser treatment

One of the important issues in PTT using inorganic nanomaterials is the toxicity of
the nanomaterials to the organs of human body. We investigated the toxicity of PSi
on the organs of mouse bodies by an indirect method of measuring the change in weight
after laser treatment. It is widely accepted that the animals given toxic treatment
lose weight. The body weight of the mouse treated with PSi followed by laser treatment
increased slightly in a pattern similar to the normal mouse without tumors (Figure
7), indicating that the mouse continued to mature without any significant toxic effect.
Another important issue in PTT using inorganic nanomaterials is harmless elimination
of the nanomaterials from the human body in a reasonable period of time. Park et al.
[31] reported that the PSi nanoparticles used for drug delivery accumulated in the organs
are noticeably cleared from the body within a period of 1 week and completely cleared
in 4 weeks. The experiments on this issue are also ongoing in our lab.

Figure 7.Change of body weight. Change in the body weight of the mice injected with a PSi/EtOH:PEG solution followed
by NIR laser treatments (4 times for 2 min at 1.5 W/cm2 each time with a time interval of 2 min). There is no significant body weight loss
for apparent side effects.

Conclusions

The in vitro cell test and in vivo animal test results were performed to investigate the feasibility of the photothermal
therapy based on PSi in combination with 808 nm NIR laser. Combination of PSi and
NIR laser treatment techniques shows a substantially higher cell death rate than only
one of these two techniques. The Murine colon carcinoma (CT-26) tumors were completely
resorbed without nearly giving damage to surrounding healthy tissue within 5 days
of PSi and NIR laser treatment. All the mice given both treatments remained healthy
and free of tumors and side effects for more than 3 months. The preliminary results
in this work shows the feasibility of photothermotherapy based on PSi in combination
with NIR laser irradiation in selectively destroying cancer cells without damaging
the surrounding healthy cells. However, the systematic administration of cancers still
remains as a challenge in this therapeutic approach. The experiments on this issue
are under way using tumor targeting techniques such as functionalization of PSi with
specific antibodies.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CL carried out the cancer photothermothrapy studies, participated in their design
and coordination, and drafted the manuscript. CH, JL, and HJ carried out the in vitro
cell tests and in vivo animal tests. SH conceived of the study, and participated in
its design and coordination. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by the Korea Engineering and Science Foundation (KOSEF) through
'the 2007 National Research Lab Program'.